A purified plasma membrane preparation of human platelets has been obtained, which is essentially free of contaminating catecholamines and nucleotides. In this preparation we have studied the regulation of the adenylate cyclase activity by alpha-adrenergic agents, PGE1, GTP and ADP. PGE1 stimulation is greatly enhanced by increasing concentrations of GTP, while alpha-adrenergic inhibition of activity is totally dependent on relatively high concentrations of GTP. ADP, a primary physiological inducer of platelet aggregation and potent inhibitor of cyclic AMP production in intact cells, has been shown to inhibit adenylate cyclase activity in this preparation with a Ki of ca. 0.5 micron M. Hitherto, such inhibition has not been demonstrated, because of generation of ADP in the adenylate cyclase assay and because of contaminating ADP in membrane preparations. Two other ADP analogs, ADP-S greater than AP(CH2)P also inhibit the enzyme.